The invention relates to polypeptides and peptides, particularly recombinant polypeptides and peptides, which can be used for the diagnosis of tuberculosis. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against tuberculosis.
It also relates to nucleic acids coding for said polypeptides and peptides.
Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against tuberculosis.
By xe2x80x9crecombinant polypeptides or peptidesxe2x80x9d it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression xe2x80x9crecombinant polypeptidesxe2x80x9d such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
The term xe2x80x9crecombinantxe2x80x9d indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host.
Nevertheless, it must be understood that this expression does not exclude the possibility for the polypeptide to be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules.
The expression xe2x80x9cbiologically purexe2x80x9d or xe2x80x9cbiological purityxe2x80x9d means on the one hand a grade of purity such that the recombinant polypeptide can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants.
Tuberculosis remains a major disease in developing countries. The situation is dramatic in some countries, particularly where high incidence of tuberculosis among AIDS patients represents a new source of dissemination of the disease.
Tuberculosis is a chronic infectious disease in which cell-mediated immune mechanisms play an essential role both for protection against and control of the disease.
Despite BCG vaccination, and some effective drugs, tuberculosis remains a major global problem. Skin testing with tuberculin PPDI (protein-purified derivative) largely used for screening of the disease is poorly specific, due to cross reactivity with other pathogenic or environmental saprophytic mycobacteria.
Moreover, tuberculin PPD when used in serological tests (ELISA) does not permit discrimination between patients who have been vaccinated by BCG, or those who have been primo-infected, from those who are developing evolutive tuberculosis and for whom an early and rapid diagnosis would be necessary.
A protein with a molecular weight of 32-Kda has already been purified from zinc deficient M. bovis BCG culture filtrate. This protein was identified as antigen 85A (De Bruyn J. et al., 1987, xe2x80x9cPurification, partial characterization and identification of a 32-KDa potein antigen of Mycobacterium bovis BCGxe2x80x9d Microb, Pathogen, 2:351). its NH2-terminal amino acid sequence (Phe-Ser-Arg-Pro-Gly-Leu; SEQ ID NO: 1) is identiacal to that reported for the xcex1-antigen (antigen 85B) protein purified from M. bovis BCG (Wiker, H. G. et al., 1986, xe2x80x9cMPB59, a widely cross-reacting protein of Mycobacterium bovis BCGxe2x80x9d Int. Arch. Allergy Appl, Immunol. 81:307). The antigen 85-complex is present among different strains of mycobacteria (De Bruyn J. et al., 1989, xe2x80x9cEffect of zinc deficiency of the appearance of two immunodominant protein antigens (32-kDa and 65-kDa) in culture filtrates of Mycobacteriaxe2x80x9d J. Gen Microbiol. 135:79). It is secreted by living bacilli as a predominant protein in normal Sauton culture filtrate and could be useful in the serodiagnosis of tuberculosis (Turneer M. et al., 1988, xe2x80x9cHumoral immune response in human tuberculosis: immunoglobulins G, A and M directed against the purified P32 protein antigen of Mycobacterium bovis bacillus Calmette-Gudrinxe2x80x9d J. Clin, Microbiol. 26:1714) and leprosy (Rumschlag H. S. et al., 1988, xe2x80x9cserological response of patients with lepromatous and tuberculosis leprosy to 30-, 31- and 32-kilodalton antigens of Mycobacterium tuberculosisxe2x80x9d J. Clin. Microbiol. 26:2200). Furthermore, the 32-kDa protein-induced specific lymphoproliferation and interferon-xcex3 (IFN-xcex3) production in peripheral blood leucocytes from tuberculosis (Huygen K, et al., 1988, xe2x80x9cSpecific lymphoproliferation, -y-interferon production and serum immunoglobulin G directed against a purified 32-kDa mycobacterial antigen (P32) in patients with active tuberculosisxe2x80x9d Scand. J. Immunol, 27:187). and leprosy patients and from PPD- and lepromin-positive healthy subjects. Recent findings indicate that the amount of 32 kDa protein induced IFN-xcex3 in BCG sensitized mouse spleen cells is under probable H-2 control (Huygen K. et al, 1989, xe2x80x9cH linked control of in vitro I interferon production in response to a 32 kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Gudrinxe2x80x9d Infect. Immo 56:3196). Finally, the high affinity of mycobacteria for fibronectin is related to proteins of the antigen 85 complex (Abou-Zeid C, et al., 1988, xe2x80x9cCharacterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCGxe2x80x9d Infect, Imm, 56:3046).
Wiker et al. (Wiker H. G. et al., 1990, xe2x80x9cEvidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complexxe2x80x9d Infect. Immun. 58:272) showed recently that the antigens 85A, B and C isolated from M. bovis BCG culture filtrate present a few amino acid replacements in their NH2 terminal region strongly suggesting the existence of multiple genes, coding for these proteins. But, the data given for the antigen 85C of M. bovis BCG are insufficient to enable its unambiguous identification as well as the characterization of its structural and functional elements.
The gene encoding the 85A antigen from Mycobacterium tuberculosis has been described (Borremans L. et al., 1989, xe2x80x9cCloning, sequence determination and expression of a 32-kilodalton protein gene of Mycobacterium tuberculosisxe2x80x9d Infect. Immun. 57:3123) which presented 77.5% homology at the DNA level within the coding region with the xcex1-antigen gene (85B gene of M. bovis BCG, substrain Tokyo) (Matsuo K. et al., 1988, xe2x80x9cCloning and expression of the Mycobacterium bovis BCG gene for extracellular xcex1-antigenxe2x80x9d J. Bacteriol. 170:3847). Moreover, recently a corresponding 32-kDa protein genomic clone from a xcexgt11 BCG library (prepared from strain M. bovis BCG 1173P2) was isolated and sequenced. The complete sequence of this gene is identical with that from the 85A gene of Mycobacterium tuberculosis except for a single silent nucleotlide change (De Wit L. et al., 1990, xe2x80x9cNucleotide sequence of the 32 kda-protein gene (antigen 85A) of Mycobacterium bovis BCGxe2x80x9d Nucl. Ac. Res. 18:3995). Thus, it was likely, but not demonstrated, that the genome of M. bovis BCG contained at least two genes coding for antigen 85A and 85B respectively. As to the genome of the Mycobacterium tuberculosis and M. bovis, nothing was proved as to the existence of new genes, besides the genes coding respectively for 85A and 85B.
An aspect of the invention is to provide a new family of nucleic acids coding for new proteins and polypeptides which can be used for the detection and control of tuberculosis.
Another aspect of the invention is to provide nucleic acids coding for the peptidic chains of biologically pure recombinant polypeptides which enable their preparation on a large scale.
Another aspect of the invention is to provide antigens which can be used
in serological tests as an in vitro rapid diagnostic test for tuberculosis or in skin test,
or as immunogenic principle of a vaccine.
Another aspect of the invention is to provide a rapid in vitro diagnostic means for tuberculosis, enabling it to discriminate between patients suffering from an evolutive tuberculosis from those who have been vaccinated against BCG or who have been primo-infected.
Another aspect of the invention is to provide nucleic probes which can be used as in vitro diagnostic reagents for tuberculosis as well as in vitro diagnostic reagents for identifying M. tuberculosis from other strains of mycobacteria.
The nucleic acids of the invention
contain a nucleotide sequence.extending from the extremity constituted by the nucleotide at position (1) to the extremity constituted by the nucleotide at position (149) represented on FIG. 1,
or contain one at least of the nucleotide sequences coding for the following peptides or polypeptides,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1, or
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1, or
SQSKGQNY (SEQ ID NO: 4), or
PMVQIPRLVA (SEQ ID NO: 5), or
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6), or
PPAAPAAPAA (SEQ ID NO: 7),
or contain nucleotidic sequences:
hybridizing with the above-mentioned nucleotide sequences, or their complements,
complementary to the above-mentioned nucleotide sequences, or
which are the above-mentioned nucleotide sequences wherein T can be replaced by U,
or are constituted by the above-mentioned nucleotide sequences.
SQSNGQNY (SEQ ID NO: 4) is a sequence corresponding to the one extending from position 84 to position 91 of 85C sequence represented on FIG. 1.
PMVQIPRLVA (SEQ ID NO: 5) is a sequence corresponding to the one extending from position 191 to position 200 of 85C sequence represented on FIG. 1.
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6) is a sequence corresponding to the one extending from position 229 to position 250 of 85C sequence represented on FIG. 1.
PPAAPAAPAA (SEQ ID NO: 7) is a sequence corresponding to the one extending from position 285 to position 294 of 85C sequence represented on FIG. 1.
The hybridization takes place under the following conditions:
hybridization and wash medium:
a preferred hybridization medium contains about 3xc3x97SSC [SSC=0*15 M sodium chloride, 0.015 M sodium citrate, pH 7] about 25 mM of phosphate buffer pH 7.1, and 20% deionized formamide, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinylpyrrolidone and about 0.1 mg/ml sheared denatured salmon sperm DNA,
a preferred wash medium contains about 3xc3x97SSC, about 25 mM phosphate buffer, pH 7.1 and 20% deionized formamide;
hybridization temperature (HT) and wash temperature (WT) are between 45xc2x0 C. and 65xc2x0 C.;
for the nucleotide sequence extending from the extremity constituted by the nucleotide at position (1) to the extremity constituted by the nucleotide at position (149) represented on FIG. 1:
HT=WT=65xc2x0 C.
for the nucleic acids of the invention defined by coded polypeptides X-Y: i.e.
the sequence extending from the extremity constituted by the amino acid at position (X) to the extremity constituted by the amino acid at position (Y) represented on FIG. 1,
the sequence extending from the extremity constituted by the amino acid at position (xe2x88x9246) to the extremity constituted by the amino acid at position (xe2x88x921) represented on FIG. 1,
HT=WT=65xc2x0 C.
the sequence extending from the extremity constituted by the amino acid at position (xe2x88x9221) to the extremity constituted by the amino acid at position (xe2x88x921) represented on FIG. 1,
HT=WT=60xc2x0 C.
for the nucleic acids defined by coded polypeptides represented by their sequence,
SQSNGQNY (SEQ ID NO: 4) HT=WT=45xc2x0 C.
PMVQIPRLVA (SEQ ID NO: 5) HT=WT=55xc2x0 C.
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6) HT=WT=65xc2x0 C.
PPAAPAAPAA (SEQ ID NO: 7) HT=WT=65xc2x0 C.
The above-mentioned temperatures are to be expressed as approximately xc2x15xc2x0 C.
Advantageous nucleic acids of the invention contain at least one of the following nucleotide sequences:
the one extending from the extremity constituted by the nucleotide at position (150) to the extremity constituted by the nucleotide at position (287) on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (224) to the extremity constituted by the nucleotide at position (287) on FIG. 1,
the one extending from the extremity constituted by the, nucleotide at position (537) to the extremity constituted by the nucleotide at position (560) on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (858) to the extremity constituted by the nucleotide at position (887) on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (972) to the extremity constituted by the nucleotide at position (1037) on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (1140) to the extremity constituted by the nucleotide at position (1169) on FIG. 1,
or contain nucleotidic sequences:
hybridizing with the above-mentioned nucleotide sequences, or
complementary to the above-mentioned nucleotide sequences, or
which are the above-mentioned nucleotide sequences wherein T can be replaced by U,
or are constituted by the above-mentioned nucleotide sequences.
The hybridization takes place under the following conditions:
hybridization and wash medium are as defined above;
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by X-Y: i.e. by the sequence extending from the extremity constituted by the nucleotide at position (X) to the extremity constituted by the nucleotide at position (Y) represented on FIG. 1:
(150)-(287) HT=WT=65xc2x0 C.
(224)-(287) HT=WT=60xc2x0 C.
(537)-(560) HT=WT=45xc2x0 C.
(858)-(887) HT=WT=55xc2x0 C.
(972)-(1037) HT=WT=65xc2x0 C.
(1140)-(1169) HT=WT=65xc2x0 C.
An advantageous group of nucleic acids of the invention contains the nucleotide sequence coding for the following peptide:
SQSNGQNY (SEQ ID NO: 4)
and possibly containing the nucleotide sequence coding for the following peptide:
FSRPGLPVEYLQVP (SEQ ID NO: 8)
and liable to hybridize with the following nucleotide sequence:
CGGCTGGGAC(or T)ATCAACACCCCGGC (SEQ ID NO: 9)
and liable to hybridize neither with
GCCTGCGGCAAGGCCGGTTGCCAG (SEQ ID NO: 10)
nor with
GCCTGCGGTAAGGCTGGCTGCCAG (SEQ ID NO: 11)
nor with
GCCTGCGGCAAGGCCGGCTGCACG (SEQ ID NO: 12)
or are constituted by the above-mentioned hybridizing nucleotide sequences.
The above-mentioned hybridization can take place when the hybridization and wash medium is as indicated above; and the hybridization and wash temperature is 52xc2x0 C.
The expression xe2x80x9cnot liable to hybridize withxe2x80x9d means that the nucleic acid molecule of the invention does not contain a stretch of nucleotide hybridizing at 52xc2x0 C. in the above defined medium with the three probes defined above.
The recognition of the above-mentioned sequence of the 294 amino acids (or of the polypeptides of the invention) by the abovesaid antibodies means that the abovesaid sequence forms a complex with one of the above-mentioned antibodies.
Forming a complex between the antigen (i.e. the sequence of 294 amino acids or any polypeptide of the invention) and the antibodies and detecting the existence of a formed complex can be done according to classical techniques (such as the one using a tracer labeled with radioactive isotopes or an enzyme).
Hereafter is given, in a non-limitative way, a process for testing the selective reaction between the antigen and human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis.
This test is an immunoblotting (Western blotting) analysis, in the case where the polypeptides of the invention are obtained by recombinant techniques. This test can also be used for polypeptides of the invention obtained by a different preparation process. After sodium dodecyl sulfatexe2x80x94polyacrylamide gel electrophoresis, polypeptides of the invention are blotted onto nitrocellulose membranes (Hybond C. (Amersham)) as described by Towbin H. et al., 1979, xe2x80x9cElectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applicationsxe2x80x9d Proc. Natl. Acad. Sci. USA 76:4350-4354. The expression of polypeptides of the invention fused to xcex2-galactosidase in E. coli Y1089, is visualized by the binding of a polyclonal rabbit anti-antigen 85 serum (1:1,000) or by using a monoclonal anti-xcex2-galactosidase antibody (Promega). The secondary antibody (alkaline phosphatase anti-rabbit immunoglobulin G and anti-mouse alkaline phosphatase immunoglobulin G conjugates, respectively) is diluted as recommended by the supplier (Promega).
In order to identify selective recognition of polypeptides of the invention and of fusion proteins of the invention by human tuberculous sera, nitrocellulose sheets are incubated overnight with these sera (1:50) (after blocking a specific protein-binding sites). Reactive areas on the nitrocellulose sheets are revealed by incubation with peroxidase-conjugated goat anti-human immunoglobulin G antibody (Dakopatts, Copenhagen, Denmark) (1:200) for 4 h. After repeated washings, color reaction is developed by adding peroxidase substrate (xcex1-chloronaphtol) (Bio-Rad Laboratories, Richmond, Calif.) in the presence of peroxidase and hydrogen peroxide.
Advantageous nucleic acids of the invention contain or are constituted by one of the above-mentioned nucleotide sequences, contain an open reading frame and code for a mature polypeptide of about 30 to about 35 kD, and contain a sequence coding for a signal sequence.
Advantageous nucleic acids of the invention contain one at least of the nucleotide sequences coding for the following polypeptides:
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1, or
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1, or
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (294) represented on FIG. 1, or
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (294) represented on FIG. 1, or
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
or contain nucleotidic sequences:
hybridizing with the above-mentioned nucleotide sequences, or
complementary to the above-mentioned nucleotide sequences, or
which are the above-mentioned nucleotide sequences wherein T can be replaced by U,
or are constituted by the above-mentioned nucleotide sequences.
The hybridization takes place under the following conditions:
hybridization and wash medium are as above defined;
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined by coded polypeptides X-Y: i.e. by the coded sequence extending from the extremity constituted by the amino acid at position (X) to the extremity constituted by the amino acid at position (Y) represented on FIG. 1:
(xe2x88x9246)-(xe2x88x921) HT=WT=65xc2x0 C.
(xe2x88x9221)-(xe2x88x921) HT=WT=60xc2x0 C.
(xe2x88x9246)-(294) HT=WT=70xc2x0 C.
(xe2x88x9221)-(294) HT=WT=70xc2x0 C.
(1)-(294) HT=WT=70xc2x0 C.
Advantageous nucleic acids of the invention contain one at least of the following nucleotide sequences:
the one extending from the extremity constituted by the nucleotide at position (150) to the extremity constituted by the nucleotide at position (287) represented on FIG. 1, or
the one extending from the extremity constituted by the nucleotide at position (224) to the extremity constituted by the nucleotide at position (287) represented on FIG. 1, or
the one extending from the extremity constituted by the nucleotide at position (1) to the extremity constituted by the nucleotide at position (1169) represented on FIG. 1, or
the one extending from the extremity constituted by the nucleotide at position (150) to the extremity constituted by the nucleotide at position (1169) represented on FIG. 1, or
the one extending from the extremity constituted by the nucleotide at position (224) to the extremity constituted by the nucleotide at position (1169) represented on FIG. 1, or
the one extending from the extremity constituted by the nucleotide at position (288) to the extremity constituted by the nucleotide at position (1169) represented on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (1) to the extremity constituted by the nucleotide at position (1211) represented on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (150) to the extremity constituted by the nucleotide at position (1211) represented on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (224) to the extremity constituted by the nucleotide at position (1211) represented on FIG. 1,
the one extending from the extremity constituted by the nucleotide at position (288) to the extremity constituted by the nucleotide at position (1211) represented on FIG. 1,
or contain nucleotidic sequences:
hybridizing with the above-mentioned nucleotide sequences, or
complementary to the above-mentioned nucleotide sequences, or
which are the above-mentioned nucleotide sequences wherein T can be replaced by U,
or are constituted by one at least of the following nucleotide sequences.
The hybridization takes place under the following conditions:
hybridization and wash medium are as above defined;
hybridization temperature (HT) and wash temperature (WT) for the nucleic acids of the invention defined for the nucleic acids of the invention defined by X-Y: i.e. by the sequence extending from the extremity constituted by the nucleotide at position (X) to the extremity constituted by the nucleotide at position (Y) represented on FIG. 1:
(150)-(287) HT=WT=65xc2x0 C.
(224)-(287) HT=WT=60xc2x0 C.
(150)-(1169) HT=WT=70xc2x0 C.
(1)-(1169) HT=WT=70xc2x0 C.
(224)-(1169) HT=WT=70xc2x0 C.
(288)-(1169) HT=WT=70xc2x0 C.
The invention relates also to the polypeptides coded by the nucleic acids of the invention above defined.
Advantageous polypeptides of the invention contain at least one of the following amino acid sequences in their polypeptide chain:
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1,
or the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1, or
SQSNGQNY (SEQ ID NO: 4), or
PMVQIPRLVA (SEQ ID NO: 5), or
GLTLRTNQTFRDTYAADQGRNG (SEQ ID NO: 6), or
PPAAPAAPAA (SEQ ID NO: 7),
or are constituted by the above-mentioned polypeptide sequences.
The invention also relates to polypeptides containing, in their polypeptide chain, the following amino acid sequence,
SQSNGQNY (SEQ ID NO: 4)
and possibly the amino acid sequence
GWDINTPA (SEQ ID NO: 13)
and possibly the amino acid sequence
FSRPGLPVEYLQVP (SEQ ID NO: 8)
and containing not the amino acid sequence
ACGKAGCQ (SEQ ID NO: 14)
and not the amino acid sequence
ACGKAGCT (SEQ ID NO: 15)
Advantageous polypeptides of the invention contain in their polypeptide chain the following amino acid sequences:
SQSNGQNY (SEQ ID NO: 4)
GWDINTPA (SEQ ID NO: 13)
FSRPGLPVEYLQVP (SEQ ID NO: 8)
and one at least of the following amino acid sequences:
PMVQIPRLVA (SEQ ID NO: 5),
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6),
PPAAPAAPAA (SEQ ID NO: 7),
and containing not the amino acid sequence
ACGKAGCQ (SEQ ID NO: 14)
and not the amino acid sequence
ACGKAGCT (SEQ ID NO: 15).
The following polypeptides are new:
SQSNGQNY (SEQ ID NO: 4),
PMVQIPRLVA (SEQ ID NO: 5),
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6),
PPAAPAAPAA (SEQ ID NO: 7).
Advantageous polypeptides of the invention are the ones constituted by:
SQSNGQNY (SEQ ID NO: 4),
PMVQIPRLVA (SEQ ID NO: 5),
GLTLRTNQTFRDTYAADGGRNG (SEQ ID NO: 6),
PPAAPAAPAA (SEQ ID NO: 7),
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1.
The invention also includes the peptidic sequences resulting from the modification by substitution and/or by addition and/or by deletion of one or several amino acids in the above defined polypeptides and peptides in so far as this modification does not alter the following properties:
selective reaction with human sera from tuberculosis patients and particularly patients developing an evolutive tuberculosis,
and/or reaction with antibodies raised against the amino acid sequence extending from the extremity constituted by amino acid at position (1), to the extremity constituted by amino acid at position (294) represented on FIG. 1.
Advantageous polypeptides of the invention contain or are constituted by one of the above-mentioned polypeptide sequences, and are about 30 to about 35 kD and are preceded by a signal peptide.
Advantageous polypeptides of the invention contain in their polypeptide chain, one at least of the following amino acid sequences or are constituted by one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1.
It goes without saying that the free reactive functions which are present in some of the amino acids, which are part of the constitution of the polypeptides of the invention, particularly the free carboxyl groups which are carried by the groups Glu or Asp or by the C-terminal amino acid on the one hand and/or the free NH2 groups carried by the N-terminal amino acid or by amino acid inside the peptidic chain, for instance Lys, on the other hand, can be modified insofar as this modification does not alter the above-mentioned properties of the polypeptide.
The molecules which are thus modified are naturally part of the invention. The above-mentioned carboxyl groups can be acylated or esterified.
Other modifications are also part of the invention. Particularly, the amine or ester functions or both of terminal amino acids can be themselves involved in, the bond with other amino acids. For instance, the N-terminal amino acid can be linked to a sequence comprising from 1 to several amino acids corresponding to a part of the C-terminal region of another peptide.
The polypeptides according to the invention can be glycosylated or not, particularly in some of their glycosylation sites of the type Asn-X-Ser or Asn-X-Thr, X representing any amino acid.
Other advantageous polypeptides of the invention consist in one of the following amino acid sequences:
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1,
or the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1.
These polypeptides can be used as signal peptides, the role of which is to initiate the translocation of a protein from its site of synthesis to the membrane and which is excised during translocation.
Advantageous polypeptides of the invention are the ones constituted by:
SQSNGQNY,
PMVQIPRLVA,
GLTLRTNQTFRDTYAADGGRNG,
PPAAPAAPAA,
the one extending from the extremity constituted by amino acid at position (1) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (294) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9246) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1,
the one extending from the extremity constituted by amino acid at position (xe2x88x9221) to the extremity constituted by amino acid at position (xe2x88x921) represented on FIG. 1.
All these polypeptides are new.
Other interesting polypeptides, which are common to the already known sequences of 85A., 85B and 85C of M. tuberculosis, M. bovis and M. kansasii are (see FIG. 2A)
GWDINTPA (SEQ ID NO: 13), and
FSRPGLPVEYLQVP (SEQ ID NO: 8).
It is to be noted that the above-mentioned polypeptides are derived from the expression products of a DNA derived, as explained hereafter in the examples,
from the nucleotide sequence coding for a protein of 33-kDa secreted by Mycobacterium tuberculosis or
from the partial nucleotide sequence coding for a protein of 33-kDa secreted by M. bovis BCG, or
from related nucleotide sequences which will be hereafter designated by 85C genes.
The invention also relates to the amino acid sequences constituted by the above-mentioned polypeptides and a protein or an heterologous sequence with respect to said polypeptide, said protein or heterologous sequence comprising for instance from about 1 to about 1000 amino acids. These amino acid sequences will be called fusion proteins.
In an advantageous fusion protein of the invention, the heterologous protein is xcex2-galactosidase.
The invention also relates to any recombinant nucleic acids containing at least one of the nucleic acids of the invention inserted in a heterologous nucleic acid.
The invention relates more particularly to recombinant nucleic acid such as defined, in which the nucleotide sequence of the invention is preceded by a promoter (particularly an inducible promoter) under the control of which the transcription of said sequence is liable to be processed and possibly followed by a sequence coding for transcription termination signals.
The invention also relates to the recombinant nucleic acids in which the nucleic acid sequences coding for the polypeptide of the invention and possibly the signal peptide, are recombined with control elements which are heterologous with respect to the ones to which they are normally associated with in the mycobacterial genome, more particularly, the regulation elements adapted to control their expression in the cellular host which has been chosen for their production.
The invention also relates to recombinant vectors, particularly for cloning and/or expression, comprising a vector sequence, notably of the type plasmid, cosmid or phage DNA or virus DNA, and a recombinant nucleic acid of the invention, in one of the non-essential sites for its replication.
According to an advantageous embodiment of the invention, the recombinant vector contains, in one of its non-essential sites for its replication, necessary elements to promote the expression of polypeptides according to the invention in a cellular host and notably a promoter recognized by the RNA polymerase of the cellular host, particularly an inducible promoter and possibly a sequence coding for transcription termination signals and possibly a signal sequence and/or an anchor sequence.
According to another additional embodiment of the invention, the recombinant vector contains the elements enabling the expression by E. coli of a nucleic acid according to the invention inserted in the vector, and particularly the elements enabling the expression of the gene or part thereof of xcex2-galactosidase.
The invention also relates to a cellular host which is transformed by a recombinant vector according to the invention, and containing the regulation elements enabling the expression of the nucleotide sequence coding for the polypeptide according to the invention in this host.
The invention also relates to a cellular host chosen from among bacteria such as E. coli, transformed by a vector as defined above, or chosen from among eukaryotic organism, such as CHO cells or insect cells, transfected by a vector as above defined.
The invention relates to an expression product of a nucleic acid expressed by a transformed cellular host according to the invention.
The invention also relates to the use of any secreted polypeptide of the invention as a carrier antigen for foreign epitopes (epitopes of a polypeptide sequence heterologous with respect to the polypeptides of the invention) in the Mycobacterium bovis BCG vaccine strain.
The Mycobacterium bovis BCG vaccine strain used can be available from Institut Pasteur (Paris), under 1173P2.
The recombinant DNA comprising the nucleic acid coding for anyone of the polypeptides of the invention and the nucleic acid coding for any foreign epitopes as defined above, can contain the promoter sequence of said polypeptide of the invention, the signal sequence of said polypeptide, possibly the coding part of said polypeptide and the coding nucleic acid of the foreign epitope, said nucleic acid of the foreign epitope being for instance
either directly located after the signal sequence, and if the coding part of the polypeptide of the invention is present, upstream from the coding part of the polypeptide of the invention,
or located downstream from the coding part of the polypeptide of the invention,
or located within the coding part of the polypeptide of the invention.
The recombinant DNA as above defined can be transformed into the vaccine strain BCG where it leads to the expression and secretion of a recombinant protein antigen.
From the nucleic acids of the invention, probes (i.e. cloned or synthetic oligonucleotides) can be inferred.
These probes can be from 15 to the maximum number of nucleotides of the selected nucleic acids. The oligonucleotides can also be used either as amplification primers in the PCR technique (PCR, Mullis and Faloona, Methods in Enzymology, vol. 155, p. 335, 1987) to generate specific enzymatically amplified fragments and/or as probes to detect fragments amplified between bracketing oligonucleotide primers.
The specificity of a PCR-assisted hybridization assay can be controlled at different levels.
The amplification process or the detection process or both can be specific. The latter case giving the higher specificity is preferred.
The invention also relates to a process for preparing a polypeptide according to the invention comprising the following steps:
the culture in an appropriate medium of a cellular host which has previously been transformed by an appropriate vector containing a nucleic acid according to the invention,
the recovery of the polypeptide produced by the abovesaid transformed cellular host from the abovesaid culture, and
the purification of the polypeptide produced, eventually by means of immobilized metal ion affinity chromatography (IMAC).
The polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis.
The synthesis can be carried out in homogeneous solution or in solid phase.
For instance, the synthesis technique in homogeneous solution which can be used is the one described by Houbenweyl in the book entitled xe2x80x9cMethode der organischen chemiexe2x80x9d (Method of organic chemistry) edited by E. Wunsh, vol. 15-I et II. THIEME, Stuttgart 1974.
The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled xe2x80x9cSolid phase peptide synthesisxe2x80x9d (IRL Press, Oxford, New York, Tokyo, 1989).
The invention also relates to a process for preparing the nucleic acids according to the invention.
A suitable method for chemically preparing the single-stranded nucleic acids (containing at most 100 nucleotides of the invention) comprises the following steps:
DNA synthesis using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986.
In the case of single-stranded DNA, the material which is obtained at the end of the DNA synthesis can be used as such.
A suitable method for chemically preparing the double-stranded nucleic acids (containing at most 100 bp of the invention) comprises the following steps:
DNA synthesis of one sense oligonucleotide using the automatic xcex2-cyanoethyl phosphoramidite method described in Bioorganic Chemistry 4; 274-325, 1986, and DNA synthesis of one anti-sense oligonucleotide using said above-mentioned automatic xcex2-cyanoethyl phosphoramidite method,
combining the sense and anti-sense oligonucleotides by hybridization in order to form a DNA duplex,
cloning the DNA duplex obtained into a suitable plasmid vector and recovery of the DNA according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
A method for the chemical preparation of nucleic acids of length greater than 100 nucleotidesxe2x80x94or base pairs, in the case of double-stranded nucleic acidsxe2x80x94comprises the following steps:
assembling of chemically synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the succession of amino acids in the natural peptide, according to the principle described in Proc. Nat. Acad. Sci. USA 80; 7461-7465, 1983,
cloning the DNA thereby obtained into a suitable plasmid vector and recovery of the desired nucleic acid according to classical methods, such as restriction enzyme digestion and agarose gel electrophoresis.
The invention also relates to antibodies themselves formed against the polypeptides according to the invention.
It goes without saying that this production is not limited to polyclonal antibodies.
It also relates to any monoclonal antibody produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the purified polypeptide of the invention on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by its ability to produce the monoclonal antibodies recognizing the polypeptide which has been initially used for the immunization of the animals.
The invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type.
The peptides which are advantageously used to produce antibodies, particularly monoclonal antibodies, are the following ones listed in Table 1 (referring to FIG. 1):
The amino acid sequences are given in the one-letter code.
Variations of the peptides listed in Table 1 are also possible depending on their intended use. For example, if the peptides are to be used to raise antisera, the peptides may be synthesized with an extra cysteine residue added. This extra cysteine residue is preferably added to the amino terminus and facilitates the coupling of the peptide to a carrier protein which is necessary to render the small peptide immunogenic. If the peptide is to be labeled for use in radioimmunoassays, it may be advantageous to synthesize the protein with a tyrosine attached to either the amino or carboxyl terminus to facilitate iodination. These peptides therefore possess the primary sequence of the peptides listed in Table 1 but with additional amino acids which do not appear in the primary sequence of the protein and whose sole function is to confer the desired chemical properties to the peptides.
The invention also relates to any polypeptide according to the invention labeled by an appropriate label of the enzymatic, fluorescent, radioactive type.
The invention also relates to a process for detecting in vitro antibodies related to tuberculosis in a human biological sample liable to contain them, this process comprising
contacting the biological sample with a polypeptide or a peptide according to the invention under conditions enabling an in vitro immunological reaction between said polypeptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by a human serum.
The detection can be carried out according to any classical process.
By way of example, a preferred method brings into play an immunoenzymatic process according to an ELISA, immunofluorescent, or radioimmunological (RIA) technique, or the equivalent ones.
Such a method for detecting in vitro antibodies related to tuberculosis comprises for instance the following steps:
deposit of determined amounts of a polypeptidic composition according to the invention in the wells of a titration microplate,
introduction into said wells of increasing dilutions of the serum to be diagnosed,
incubation of the microplate,
repeated rinsing of the microplate,
introduction into the wells of the microplate of labeled antibodies against the blood immunoglobulins,
the labeling of these antibodies being based on the activity of an enzyme which is selected from among the ones which are able to hydrolyze a substrate by modifying the absorption of the radiation of this latter at least at a given wavelength,
detection by comparison with a control standard of the amount of hydrolyzed substrate.
The invention also relates to a process for detecting and identifying in vitro antigens of M. tuberculosis in a human biological sample liable to contain them, this process comprising:
contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. tuberculosis which are possibly present in the biological sample and the in vitro detection of the antigen/antibody complex which may be formed.
Preferably, the biological medium is constituted by sputum, pleural effusion liquid, broncho-alveolar washing liquid, urine, biopsy or autopsy material.
The invention also relates to an additional method for the in vitro diagnosis of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising the following steps:
the possible previous amplification of the amount of the nucleotide sequences according to the invention, liable to be contained in a biological sample taken from said patient by means of a DNA primer set as defined above,
contacting the above-mentioned biological sample with a nucleotide probe of the invention, under conditions enabling the production of an hybridization complex formed between said probe and said nucleotide sequence,
detecting the abovesaid hybridization complex which has possibly been formed.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis as defined above, the following necessary or kit can be used, with said necessary or kit comprising:
a determined amount of a nucleotide probe of the invention,
advantageously the appropriate medium for creating an hybridization reaction between the sequence to be detected and the above mentioned probe,
advantageously, reagents enabling the detection of the hybridization complex which has been formed between the nucleotide sequence and the probe during the hybridization reaction.
The invention also relates to an additional method for the in vitro diagnosis of tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis comprising:
contacting a biological sample taken from a patient with a polypeptide or a peptide of the invention, under conditions enabling an in vitro immunological reaction between said polypeptide or peptide and the antibodies which are possibly present in the biological sample and
the in vitro detection of the antigen/antibody complex which,has possibly been formed.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis, the following necessary or kit can be used, with said necessary or kit comprising:
a polypeptide or a peptide according to the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling to detect the antigen/antibody complex which has been produced by the immunological reaction, with said reagents possibly having a label, or being liable to be recognized by a labeled reagent, more particularly in the case where the above-mentioned polypeptide or peptide is not labeled.
The invention also relates to an additional method for the in vitro diagnosis of tuberculosis in a patient liable to be infected by M. tuberculosis, comprising the following steps:
contacting the biological sample with an appropriate antibody of the invention under conditions enabling an in vitro immunological reaction between said antibody and the antigens of M. tuberculosis which are possibly present in the biological sample and the in vitro detection of the antigen/antibody complex which may be formed.
To carry out the in vitro diagnostic method for tuberculosis in a patient liable to be infected by Mycobacterium tuberculosis, the following necessary or kit can be used, with said necessary or kit comprising:
an antibody of the invention,
reagents for making a medium appropriate for the immunological reaction to occur,
reagents enabling the detection of the antigen/antibody complexes which have been produced by the immunological reaction, with said reagent possibly having a label or being liable to be recognized by a labeled reagent, more particularly in the case where the above-mentioned antibody is not labeled.
An advantageous kit for the in vitro diagnosis of tuberculosis comprises:
at least a suitable solid phase system, e.g. a microtiter-plate for deposition thereon of the biological sample to be diagnosed in vitro,
a preparation containing one of the monoclonal antibodies of the invention,
a specific detection system for said monoclonal antibody,
appropriate buffer solutions for carrying out the immunological reaction between the biological sample and said monoclonal antibody on the one hand, and the bonded monoclonal antibodies and the detection system on the other hand.
The invention also relates to a kit, as described above, also containing a preparation of one of the polypeptides or peptides of the invention, with said antigen of the invention being either a standard (for quantitative determination of the antigen of M. tuberculosis which is sought) or a competitor, with respect to the antigen which is sought, for the kit to be used in a competition dosage process.
The invention also relates to a necessary or kit for the diagnosis of prior exposure of a subject to M. tuberculosis, with said necessary or kit containing a preparation of at least one of the polypeptides or peptides of the invention, with said preparation being able to induce in vivo, after being intradermally injected to a subject, a delayed-type hypersensitivity reaction at the site of injection, in case the subject has had prior exposure to M. tuberculosis. 
This necessary or kit is called a skin test.
The invention also relates to an immunogenic composition comprising a polypeptide or a peptide according to the invention, in association with a pharmaceutically acceptable vehicle.
The invention also relates to a vaccine composition comprising among other immunogenic principles any one of the polypeptides or peptides of the invention or the expression product of the invention, possibly coupled to a natural protein or to a synthetic polypeptide having a sufficient molecular weight so that the conjugate is able to induce in vivo the production of antibodies neutralizing Mycobacterium tuberculosis, or induce in vivo a cellular immune response by activating M. tuberculosis antigen-responsive T cells.
The peptides of the invention which are advantageously used as immunogenic principle are the ones mentioned in Table 1.
Other characteristics and advantages of the invention will appear in the following examples and the figures illustrating the invention.